The effect of dextrose prolotherapy on patients diagnosed with knee osteoarthritis: A comprehensive systematic review and meta‐analysis of interventional studies

Abstract Background and Aims The primary objective of this systematic review and meta‐analysis was to assess the impact of dextrose prolotherapy on individuals diagnosed with knee osteoarthritis (KOA). Methods To conduct a thorough investigation, a variety of leading international databases were checked, including PubMed (Medline), Scopus, Web of Sciences, EMBASE (Elsevier), ClinicalTrials.gov, and the Cochrane Library. The search covered a period from January 2000 to the end of June 2023, which facilitated the collection of relevant studies. Results The findings of the study revealed that when the studies utilizing the Western Ontario McMaster Universities Index tool (WOMAC) were combined, patients with KOA who received prolotherapy experienced an improvement in function compared with those who received other treatments (SMD: 0.20; 95% Confidence Interval [1]: −0.11, 0.51; p value SMD = 0.221; I 2: 78.49%; p heterogeneity < 0.001). Additionally, there was a decrease in mean pain and stiffness among patients who received prolotherapy compared with those who received other treatments or a placebo [(SMD: −0.95; 95% CI: ‐1.14, −0.76; p value SMD < 0.001; I 2: 59.35%; p heterogeneity = 0.070) and (SMD: −0.21; 95% CI: −0.32, −0.10; p value SMD < 0.001; I 2: 88.11%; p heterogeneity < 0.001)]. Furthermore, based on the Visual Analog Scale (VAS) score, there was a reduction of 0.81 units out of 10 in mean pain for patients with KOA who received prolotherapy (SMD: −0.81; 95% CI: −5.63, 4.10; p value SMD = 0.693; I 2: 48.54%; p heterogeneity = 0.08). Conclusion Drawing from the data analysis performed in this meta‐analysis, it is apparent that dextrose prolotherapy exhibits promising effectiveness in reducing joint pain and stiffness, as well as improving functional performance in individuals suffering from KOA. Furthermore, it is recommended that forthcoming studies incorporate follow‐up periods to guide decisions concerning the duration of prolotherapy's effects.


| INTRODUCTION
Osteoarthritis (OA) is the most common form of arthritis, posing a serious public health concern worldwide, with an increase in disability-adjusted life years (DALY) in most countries.This cost is mostly caused by demographic shifts toward older populations and the rising prevalence of obesity.In 2017, globally, the agestandardized point prevalence and annual incidence rate of OA were recorded at 3754.2 and 181.2 per 100,000 individuals, respectively. 1,2OA is a multifactorial disease characterized by the progressive degeneration of joint components, particularly articular cartilage. 3The pathophysiology of OA is complex, involving inflammatory processes, epigenetic control, cell death pathways, and the synovial lymphatic system.Danger-associated molecular patterns (DAMPs) and immune system activation both trigger inflammatory responses within the joint, resulting in tissue damage and inflammation.Epigenetic changes, such as DNA methylation and microRNA regulation, influence gene expression patterns involved in cartilage degradation.Joint tissue degradation is caused by a variety of cell death mechanisms, including pyroptosis and autophagy.6][7] KOA refers to structural changes in the knee joint that affect the articular cartilage, the subchondral bone, and other joint structures.Age, weight, heredity, and gender are all risk factors for developing KOA.
KOA etiology includes a reduction in cartilage repair mechanisms with age, increased mechanical stress on joints due to obesity, a hereditary susceptibility to joint degeneration, and gender differences in joint biomechanics.5][16] The use of injection techniques (one of the treatment modalities) in the treatment of KOA, including prolotherapy, is a topic of significant interest and ongoing research.Among the various substances used in injections for KOA, such as steroid injections, hyaluronic acid (HA), platelet-rich plasma (PRP) injections, and prolotherapy, prolotherapy stands out as a promising alternative.Prolotherapy involves the injection of an irritant solution, typically dextrose, to stimulate the body's natural healing processes and promote tissue repair in the knee joint.8][19] The comprehensive review of literature on prolotherapy highlights its potential benefits in managing OA symptoms, particularly in the knee joint.1][22][23][24][25] Dextrose, a cost-effective and readily available substance, has gained prominence as an injection-based therapeutic option for managing chronic painful musculoskeletal conditions.
Prolotherapy, which has been in use for decades, entails the injection of dextrose or other proliferants into affected tissues.
The surge in interest in prolotherapy during the 1990s among both physicians and patients can be attributed to its proposed mechanism of action, centered around tissue repair and regeneration, ultimately leading to pain alleviation and enhanced functional outcomes.7][28][29] Despite extensive clinical trial investigations worldwide, the impact of prolotherapy on KOA remains contentious, with studies yielding conflicting results. 23,30Consequently, treatment guidelines for KOA seldom endorse the use of prolotherapy, and when mentioned, it is often subject to specific conditions.Notably, guidelines from the American College of Rheumatology/Arthritis Foundation and the Osteoarthritis Research Society International (OARSI) cautiously recommend the use of dextrose prolotherapy for KOA treatment.The objective of this study is to evaluate the effect of dextrose prolotherapy on KOA patients through a systematic review and meta-analysis approach.

| METHODS
The current study followed a systematic review and meta-analysis approach, which involved six key steps: search syntax and search strategy, screening, selection, data extraction, risk of bias assessment, and meta-analysis.The study adhered to the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) to ensure the systematic review and metaanalysis were conducted and reported in a transparent and consistent manner. 31

| Search strategy and screening
The search for relevant studies in this research was conducted in several international databases, including PubMed (Medline), Scopus, Web of Sciences, EMBASE (Elsevier), ClinicalTrials.gov,and Cochrane Library.The search period spanned from January 2000 to the end of July 2023.The main keywords used in the search included "Prolotherapy," "Dextrose Prolotherapy," "Knee Osteoarthritis," and "Knee Arthrosis."Thesauruses, Emtree, and Mesh were consulted to find synonyms related to these keywords.The search syntaxes for each database were formulated using "AND" and "OR" operators based on the chosen synonyms.Additionally, a manual search was conducted by examining the reference lists of relevant articles to ensure comprehensive coverage.After retrieving the search results, the articles were imported into Endnote software version 9.
Duplicate articles were identified and excluded based on matching titles, authors, and publication years.The remaining articles were then screened based on their title, abstract, and full text.The inclusion criteria for this meta-analysis were carefully applied, and only studies that adhered to the PICOT structure (as specified in Table 1) were included.Studies that did not provide the required information outlined in Table 1 were excluded from the analysis.All aspects of the search strategy and article screening were independently performed by two authors (SKH and LM/YM).

| Data extraction
During this stage of the study, the following information was extracted for the meta-analysis: authors' names, publication year, country of origin, sample size, mean age of participants, body mass index (BMI), disease duration, duration of the follow-up period after the intervention, tools used to measure pain, characteristics of the studied population, method and type of intervention, and mean pain levels before and after the intervention.The data extraction process was carried out independently by two authors (S.K. H. and L. M.).In case of any disagreements, a third person was involved to resolve them and ensure consensus was reached on the extracted data (Y.M.).

| Quality assessment
The quality of the study design, sampling strategy, and measurement was evaluated using the revised Cochrane risk-of-bias tool for randomized trials (RoB 2).For studies employing a cross-over design, special considerations were taken into account based on the Cochrane Handbook of Systematic Reviews. 32Two authors independently assessed the risk of bias within each trial using Revman 5.3 software (S.K. H. and M. A./A. M.).In the event of any disagreements, consensus was reached through discussion between the two authors.If a persistent disagreement arose, a third party (Y. M.) was consulted to resolve the disagreement.
T A B L E 1 The criteria for inclusion of studies in the present meta-analysis.
Population (P) Type of study (T) People with knee osteoarthritis (KOA) were the target population for this meta-analysis.The desired intervention in the meta-analysis discussed in the sources was dextrose prolotherapy with varying concentrations administered to patients with knee osteoarthritis.
The comparator group included other treatments or placebo.
Pain or its mean based on the WOMAC, VAS, and KOSS tools was the intended outcome.Various types of clinical trial studies, including randomized controlled trials (such as parallel and crossover designs), nonrandomized controlled trials, and before-after studies, are conducted to evaluate the efficacy and safety of desired intervention.

| Statistical analysis
In this study, the desired effect size was assessed using the standardized mean difference (SMD), calculated using Cohen's method.Mean pain levels in the intervention and placebo groups before and after the intervention were extracted from the primary studies.

| RESULTS
In this meta-analysis, the initial search strategy in the selected databases yielded a total of 433 studies.After removing duplicates and screening the remaining studies based on their titles, 48 studies were deemed potentially relevant.Subsequently, after screening the abstracts, 27 articles were excluded, leaving 21 studies for full-text screening.
During the full-text screening, seven studies were excluded from the meta-analysis due to disparate outcomes, two studies due to differences in intervention and desired effect size, and one study due to the unavailability of the full text.Ultimately, 11 clinical trials remained for further review and analysis in the present meta-analysis [33][34][35][36][37][38][39][40][41][42] (Figure 1).
In the clinical trials chosen, prolotherapy was the intervention employed, with varying doses of dextrose used across the trials.More precisely, four trials considered prolotherapy using 5 mL of dextrose, while one trial used 10 mL, another used 3.5 mL, one more used 2 mL, one used 6 mL, and two used 7 mL.A study did not provide specific information on the amount of dextrose used in prolotherapy.Out of the clinical trials selected, five studies included a control group using HA, two studies used a control group with PRP, three studies had a control group with normal saline, and one study included a control group with

autologous conditioned serum (ACS). The Western Ontario McMaster
Universities Index tool (WOMAC), Visual Analog Scale (VAS), and knee injury and osteoarthritis outcome score (KOOS) indexes were mainly utilized in the 11 chosen studies to evaluate pain and other results.
Regarding postintervention follow-up, two investigations assessed results at 24 weeks, two investigations at 52 weeks, two investigations at 12 weeks, one investigation at 18 weeks, and three investigations had a follow-up of 10 weeks or fewer.Table 2 offers more comprehensive information.

| Mean of WOMAC tool and its subscales
The results of the meta-analysis, combining studies that used the WOMAC tool, revealed the following findings:

| Publication bias
The results of the Egger's test indicated the presence of publication bias in the combined studies related to function based on the WOMAC tool (B: −6.22; SE: 1.39; p value < 0.001).
However, after conducting a trim and fill analysis to assess the impact of publication bias on the estimated findings, it was determined that bias did not significantly affect the calculated result (SMD: 0.205; 95% CI: −0.The prolotherapy effect on pain in patients with KOA decreased with increasing the KOA history (B: −0.17; SE: 0.11; p value > 0.05; 95% CI: −0.44, 0.06).Also, with the increase in the follow-up period after the prolotherapy intervention, its effect on the pain of patients with KOA increased (B: 0.12; SE: 0.07; p value > 0.05; 95% CI: −0.01, 0.27).
Also, the meta-regression results showed the prolotherapy effect on stiffness with the increase in the KOA history and the duration of the follow-up period after the treatment decreased (B: −0.

| Mean pain based on VAS score
In the present meta-analysis, three studies with seven effect sizes (SMD) reported the mean pain in the two groups of prolotherapy (injection of 3-9 mL of 25%-50% dextrose) and control.The two pain differences based on VAS were reported as percentages or in units of 100 while the rest of the effect sizes reported the mean pain in the range of 0 to 10.Therefore, the two effect sizes reported based on the units of 100 were excluded from the research and the rest of the studies with the reported effect sizes were combined together.The meta-analysis result showed the mean pain as an SMD decreased 0.81 units out of 10 (SMD: −0.81; 95% CI: −5.63, 4.10; p SMD > 0.05; I 2 : 48.54%; p heterogene- ity = 0.08) (Figure 3, Table 3).
F I G U R E 2 Forest plot of the effect of prolotherapy (3-9 mL with 25%-50% dextrose) on the WOMAC score in patients with knee osteoarthritis.
T A B L E 3 The mean difference and related effect size of included randomized control trials.| 11 of 17

| Publication bias
The results of the Eggers test showed the publication bias occurred in the results related to the combination of studies determining the prolotherapy effect on the mean pain (B: −19.88;SE: 4.13; p value < 0.001).
Because the number of articles was small, funnel plot was not used.
The prolotherapy effect on the mean pain in patients with KOA was evaluated based on the disease history and the duration of the follow-up period after the intervention using meta-regression.The number of studies reporting the mean history of KOA in patients was small.Therefore, meta-regression analysis was not performed for this variable, but for the follow-up duration, the results showed the prolotherapy effect on the mean pain in patients with KOA increased with the increase in the follow-up period after the intervention (B: 0.16; SE: 0.54; p value > 0.05; 95% CI: −0.89, 1.22).

| Mean of KOOS tool and its subscales
The results showed after combining the studies which used the KOOS tool, function in patients with KOA, who received prolotherapy significantly increased by 0.68 compared with other treatments (in one study with three different effect sizes) (SMD: 0.68; % 95 CI: 0.09, 1.27; p SMD < 0.05; I 2 : 51.88%; p heterogeneity > 0.05) (Figure 4).
The mean pain based on this questionnaire significantly increased by Forest plot of the effect of prolotherapy (3-9 mL with 25 injections to 50% dextrose) on the VAS score in patients with knee osteoarthritis.
F I G U R E 4 Forest plot of the effect of prolotherapy (3-9 mL with 25%-50% dextrose) on the KOOS score in patients with knee osteoarthritis.
Because the number of studies in the combination of the results of the mean KOOS tool and its scales was small, often only one study in the scale analyzes, meta-regression analysis and publication bias evaluation were not performed.

| Quality assessment results
The results of the quality assessment, conducted using the Cochrane checklist, indicated that the majority of the selected RCTs were of high quality.The results indicated that two studies were categorized as having selection and detection bias, three studies were categorized as having performance bias, and four studies were categorized as having attrition bias and selective bias.Please refer to Figure 5 for a visual representation of the quality assessment results.

| DISCUSSION
The study conducted a comprehensive systematic review and metaanalysis on the impact of prolotherapy in patients with KOA.While the precise mechanisms of prolotherapy on soft tissues and joints are not fully understood, previous research has proposed several potential mechanisms. 22,24,36,45,46One mechanism is the reduction of local inflammation, where the injection of a proliferative solution may aid in decreasing inflammation and promoting healing, potentially alleviating symptoms like pain and stiffness in KOA patients.triggered by prolotherapy is believed to promote tissue proliferation and remodeling, aiding in the healing process. 25,36,47,48  to address these challenges in human samples.This will help establish a clearer understanding of how prolotherapy affects joints and tissues, and potentially improve its effectiveness as a therapeutic approach for conditions like KOA. 52 The second hypothesis regarding the effect of dextrose in prolotherapy is that it has a prechondrogenic effect.This hypothesis suggests that dextrose may promote the formation of new cartilage.However, the available evidence on this topic is conflicting, and there is no consensus among researchers. 53r example, a systematic review highlighted the positive and significant beneficial effects of dextrose prolotherapy in the treatment of symptomatic KOA, suggesting its potential in the management of chronic musculoskeletal conditions.In addition, research has shown that dextrose solutions can stimulate the production of growth factors essential for the repair and growth of tendons, ligaments, and other soft tissues, indirectly supporting the idea of a prechondrogenic effect.It is important to note, however, that while the regenerative potential of dextrose in prolotherapy has been demonstrated, the claim of direct cartilage regeneration must be approached with caution. 50,54,55The third hypothesis regarding the effect of prolotherapy is related to its modulating effect on pain.
However, there is conflicting information regarding this hypothesis as well.The results of the present meta-analysis can contribute to reducing these contradictions and shedding light on the effect of prolotherapy on pain in KOA patients.According to the results of the meta-analysis, after combining selected clinical trial studies, there was an increase in function among KOA patients who received improving functional outcomes for KOA patients. 22,24,56e meta-regression results showed the prolotherapy effect on knee function increases with increasing age and BMI because they are two important risk factors in the occurrence of KOA.Also, the results of meta-regression in the present meta-analysis showed the prolotherapy effect on knee function increased over time after prolotherapy injection in patients.Therefore, the more time passes after prolotherapy injection in patients with KOA, their knee function improves which can be due to the increase in the strength of the ligaments.The results of the present meta-analysis showed the mean pain based on the WOMAC and VAS tools in these patients significantly decreased compared with the ones who received other treatments or placebo.In patients with KOA, pain is caused due to the destruction of the cartilages of the bone heads, the reduction of the joint space, the damage of the ligaments, and finally the Another suggested mechanism is the promotion of proliferation in joints or soft tissues, with prolotherapy solutions like dextrose stimulating tissue growth and repair, leading to improved joint function.Additionally, prolotherapy has been linked to pain reduction by modulating pain signals and providing analgesic effects through injections into the affected area.Prolotherapy works in the treatment of KOA by inducing an inflammatory response through the injection of a solution, typically containing substances like dextrose and lidocaine, near the painful or damaged joints.Dextrose, a type of sugar, can indeed induce an inflammatory response when administered, particularly in the short term.When used in prolotherapy, dextrose solutions can stimulate an inflammatory reaction that triggers the body's natural healing processes.This inflammatory response is essential for the repair and regeneration of tissues like tendons, ligaments, and cartilage.Studies have shown that dextrose F I G U R E 5 Risk of bias graph: review authors' judgments about each risk of bias item presented as percentages across and judgments about each risk of bias item for each all-included study.concentrations above 10% can operate through inflammatory mechanisms, while lower concentrations are considered noninflammatory.The inflammatory response induced by dextrose in prolotherapy is a crucial part of the treatment process, as it promotes the production of growth factors necessary for tissue repair and growth.Therefore, while dextrose may create inflammation in the short term, it plays a beneficial role in stimulating tissue healing and regeneration in the context of prolotherapy.This process initiates the production of fibroblasts, which are the body's repair cells, leading to the deposition of new tissue fibers that repair the injury, stabilize the area, restore function, and reduce pain.The inflammatory cascade Additionally, in a high glucose environment, there is an increase in the production of platelet-derived growth factors by vascular endothelial and mesangial cells.While these functions have been observed in laboratory experiments, the practical application of these findings in human samples still presents challenges.The translation of laboratory findings to clinical settings involves complexities such as the variability of patient response, the influence of other factors in the healing process, and the need for further validation in controlled clinical trials.It is important for future research to continue investigating the mechanisms of action underlying prolotherapy and prolotherapy, as assessed by the WOMAC questionnaire.Prolotherapy involves the injection of a proliferative solution into damaged or inflamed joints, which aims to stimulate and accelerate the body's natural healing process.The increased speed of the repair process can potentially lead to an improvement in knee joint function.While pain modulation is a key aspect of prolotherapy, the specific mechanisms by which prolotherapy exerts its pain-relieving effectsare not yet fully understood.It is believed that prolotherapy injections may have analgesic effects by reducing inflammation and promoting tissue healing, which can ultimately lead to a decrease in pain experienced by patients with KOA.The findings of the metaanalysis support the notion that prolotherapy can positively impact knee joint function in patients with OA.However, it is important to note that individual responses to prolotherapy may vary, and further research is needed to better understand the underlying mechanisms and optimize treatment protocols for pain modulation in KOA patients.40,53Research suggests that dextrose prolotherapy may be as effective as, or possibly more effective than, HA, PRP, and ACS in improving functional outcomes in patients with KOA.Specifically, studies have shown that dextrose prolotherapy was more effective in improving functional outcomes in patients with generalized KOA compared with alternative treatments such as saline, exercise, local corticosteroid (LC) injections, and pulsed radiofrequency (PRF) therapy.Additionally, in terms of pain relief, dextrose prolotherapy has been found to be more effective in reducing pain compared with other treatments including saline, exercise, LC injections, PRF therapy, HA injections, and physical therapy (PT).While some studies have shown promising results for HA, PRP, ACS, and exercise programs in improving functional outcomes, dextrose prolotherapy has demonstrated similar or superior efficacy in reducing pain and